Aerosolized Zika virus infection in Guinea pigs.

Zika virus (ZIKV) is primarily transmitted through mosquito bites and sexual contact, and vertical transmission of ZIKV has also been observed in humans. In addition, ZIKV infection via unknown transmission routes has been frequently reported in clinical settings. However, whether ZIKV can be transmitted via aerosol routes remains unknown. In this study, we demonstrated that aerosolized ZIKV is fully infectious in vitro and in vivo. Remarkably, intratracheal (i.t.) inoculation with aerosolized ZIKV led to rapid viremia and viral secretion in saliva, as well as robust humoral and innate immune responses in guinea pigs. Transcriptome analysis further revealed that the expression of genes related to viral processes, biological regulation and the immune response was significantly changed. Together, our results confirm that aerosolized ZIKV can result in systemic infection and induce both innate and adaptive immune responses in guinea pigs, highlighting the possibility of ZIKV transmission via aerosols.

Introduction

Zika virus (ZIKV) is a mosquito-borne flavivirus in the Flaviviridae family, which includes a large number of pathogens, such as dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV) [1]. Flaviviruses have single positive-stranded 11-kb RNA genomes in which the 5’ and 3’ untranslated regions flank a polyprotein coding region encoding three structural proteins [capsid, pre/membrane (prM), and envelope (E)] and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Although most Zika infections are asymptomatic or present with benign, self-limited symptoms, a small percentage of patients have complications, such as congenital anomalies in the developing foetus of pregnant women infected with the virus and neurological complications (Guillain‒Barré syndrome). To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection [2].

Similar to other mosquito-borne flaviviruses, ZIKV is primarily transmitted to humans through bites by mosquito vectors. However, human-to-human transmission has also been reported for ZIKV via blood transfusion, sexual contact, and transplacental transmission [3-6]. Moreover, infectious ZIKV can be present in saliva, urine, semen, and breast milk [7-10], and transmission via the skin or oronasal mucosa has been observed in humans and experimental animals [1,11,12]. Interestingly, a previous study suggested that DENV could be isolated from the upper respiratory tract of patients with dengue fever[13]. Another study also indicated that Tembusu virus (TMUV), a flavivirus related to ZIKV, could be transmitted efficiently among ducks by aerosol transmission [14]. Furthermore, JEV was isolated from the nasal secretions of pigs [15], and transmission has been demonstrated to occur between animals via aerosols [16]. However, whether ZIKV can infect and be transmitted via aerosols remains unknown.

Guinea pigs have been widely used as a well-established animal model to evaluate ZIKV infection and pathogenesis [1,17-19]. Our previous study indicated that guinea pigs were susceptible to ZIKV infection via subcutaneous or intranasal inoculation routes[1]. Herein, we further applied aerosolized ZIKV directly via intratracheal (i.t.) inoculation to the lungs of guinea pigs to assess the risk of aerosol-mediated ZIKV infection. The results indicated that aerosolized ZIKV can cause systemic infection and induce both innate and adaptive immune responses in guinea pigs.

Material and methods

Ethics statement

All animal experiments were performed according to the guidelines of the Chinese Regulations of Laboratory Animals (Ministry of Science and Technology of People’s Republic of China). All animal procedures were approved by the Animal Experiment Committee of the Laboratory Animal Center, AMMS, China (approval number: IACUC-IME-2021-010).

Cells and viruses

BHK-21 cells (#CCL-10) were purchased from ATCC and cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% foetal bovine serum (FBS), 100 U/mL penicillin and 100 g/ml streptomycin. C6/36 cells (ATCC# CRL-1660) were cultured in RPMI-1640 containing 10% FBS, 100 U/mL penicillin and 100 g/mL streptomycin. Guinea pigs in this study were inoculated with the ZIKV GZ01 strain (GenBank accession no: KU820898), which was originally isolated from a Chinese patient who returned from Venezuela in 2016 [7]. The viral stocks used in this study were prepared on a confluent monolayer of mosquito C6/36 cells, titrated in BHK-21 cells for the plaque-forming assay, and stored at −80 °C.

Aerosolization of ZIKV solution

Aerosolization of ZIKV solution was performed using a hand-held liquid aerosol pulmonary delivery device (HLAPDD) (Huironghe Company, Beijing, China) suitable for guinea pigs. The device is an aerosol generator that ejects aerosol solutions directly into the lungs and achieves precise quantification. The tip of the device was inserted into a 1.5-ml EP tube with a hole, and 50 μl of ZIKV aerosol was generated. The volume was measured with a pipette after centrifugation. The particle size of individual aerosols was measured using an aerodynamic particle sizer (APS 3321, TSI, USA) with a sampling flow rate of 5 L/min and time of 15 s. The mean mass aerodynamic diameter (MMAD) of the aerosol particles was determined by Aerosol Instrument Manager Software.

In vitro phenotypes of aerosolized ZIKV

Briefly, ZIKV aerosols were collected using a 1.5-ml EP tube and then centrifuged for 1 min at 8000 r/min for subsequent RT‒qPCR, immunofluorescence staining and plaque assays, and the replication curve, virus protein expression and virus titre were compared with those of the original solution.

For the replication curve, BHK-21 cells were transferred to 24-well plates and cultured at 37 °C with 5% CO2. Then, the liquid and aerosolized Zika virus were added to BHK-21 cells and incubated at 37 °C for 1 h. Infected cells were cultured at 37 °C with 5% CO2, and the cell supernatant was collected at 24, 48, and 72 h post-infection (hpi). The viral RNA load in the cell supernatant was determined by RT‒qPCR, and the replication curve of the liquid and aerosolized Zika virus on BHK-21 cells was plotted by GraphPad Prism 8 software.

For viral protein expression, the infected cells were fixed with methanol/acetone (v/v: 7/3) at 48 h post-infection. The cells were then treated with the anti-Zika ENV mAb (Cat No. BF-1176-56, 1:1000 diluted, BioFront Technologies) and incubated at 37 °C for 1 h. Goat anti-mouse IgG-Alexa Fluor 488 (1:200 diluted, Gene-Protein Link) was added, and the plates were incubated at 37 °C for 1 h. For cell nucleus staining, 4,6-diamidino-2-phenylindole (DAPI, 0.5 ng/μL) was added to the wells, and the plates were incubated for 5 min. An Olympus IX73 microscope was used for image acquisition.

For determination of the virus titre, BHK-21 cells were cultured in a 12-well plate for 24 h and then infected with 500 µL of 10-fold viral dilutions for 2 h at 37 °C. Viral supernatants were replaced with DMEM containing 1% low melting point agarose (Promega) and 2% FBS. At 4 days after infection, the cells were fixed with 4% formaldehyde, followed by staining with 1% crystal violet solution. Finally, all visible plaques were counted, and the final titres were calculated as plaque-forming units/mL (PFU/mL).

Aerosol distribution

The guinea pigs were sacrificed by exposure to CO2. Aniline blue solution was injected directly into the lung, and then the lung was dissected to verify the distribution of aerosols and photographed. To further verify the homogeneity and specificity of aerosol distribution in the lung, Near-infrared fluorescent Degradex® poly microspheres (Hopkinton, MA, USA) were sprayed into the lung via i.t. inoculation using a HLAPDD. Briefly, guinea pigs were anaesthetized with pentobarbital sodium by intraperitoneal injection. A special support equipped with a nylon wire was used to suspend the guinea pigs by their upper incisors at 60°. Optimal illumination of the trachea was achieved by a laryngoscope (Huironghe Company, Beijing, China). After a clear view of the trachea was obtained, the tip of the device was inserted, and 50 µl of aerosol was sprayed into the lung by depressing the syringe piston with a constant force [20]. The guinea pig was sacrificed 24 h after administration, and the organs were imaged using an IVIS Spectrum small-animal imaging system (λex: 745 nm; λem: 820 nm; exposure time: 10 s; binning factor, f-stop 2; field of view: D).

Animal experiments

Male guinea pigs (Hartley strain) weighing 280–300 g were purchased from Charles River Laboratories and inoculated with 105 PFU of ZIKV by the i.t. route using a HLAPDD. Serum and saliva samples were collected and processed at 1, 2, 3, 5 and 7 days post-infection (dpi) as described previously [1]. The viral loads of the samples were analysed by quantitative reverse transcription PCR (RT‒qPCR) and are expressed as RNA copies per millilitre. Guinea pigs were euthanized by exposure to CO2 at 1, 2, 3 and 7 dpi to isolate tissues. At 2 or 7 dpi, the viral loads of various tissues were detected, and lung tissues were collected for subsequent histopathology assays, immunofluorescence staining and RNA ISH assays as described below. Transcriptome analysis of the lungs was conducted at 1, 3 and 7 dpi. Cellular immune responses in serum were assessed at 1, 3, 5 and 7 dpi. Serum was collected at 7, 14, 21 and 28 dpi for the detection of ZIKV-specific IgG as described below.

Viral load assay

The viral RNA was extracted from 0.2 ml of saliva, the supernatants of tissues, the BioSampler collection medium and 0.1 ml of serum using a PureLink® RNA Mini Kit (Life Technology, USA) according to the manufacturer’s instructions and eluted in 60 μl of RNase-free water. RT‒qPCR was performed using the One Step PrimeScript RT‒PCR Kit (Takara, Japan) with the primers and probe as described previously [1]. RNA copies per ml or RNA copies per gram were calculated from quantitative PCR Ct values with a published method [21].

Histopathology assay

Following experimental ZIKV aerosol infection of guinea pigs, euthanasia was conducted at the indicated times. Lung tissues were fixed in 4% neutral buffered formalin and embedded in paraffin. The samples were cut into 5 μm thick sections, and after rehydration with a gradient, they were stained with haematoxylin and eosin. Images were captured using an Olympus BX51 microscope equipped with a DP72 camera. The original magnification was 20×.

Immunofluorescence staining

For immunostaining, lung tissues were fixed with 4% paraformaldehyde for 24 h at 4 °C and then dehydrated in 30% sucrose for 24 h. The slices were blocked at room temperature (RT) for 1 h in 3% bovine serum albumin, 10% FBS, and 0.2% Triton X-100 in PBS, incubated with mouse anti-ZIKV E protein antibody (BF-1176-56, BioFront, China) overnight at 4 °C, washed with 0.2% Triton X-100 in PBS (3×10 min), incubated in fluorescein isothiocyanate-conjugated goat anti-mouse IgG (ZSGB-Bio, China) at RT for 1 h, and then washed three times. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen).

RNA ISH assay

The ZIKV genomic RNA ISH assay was performed with an RNAscope kit (Advanced Cell Diagnostics, USA) according to the manufacturer’s instructions. Briefly, formalin-fixed paraffin-embedded (FFPE) tissue samples were deparaffinized with xylene and incubated with hydrogen peroxide for 10 min at room temperature to neutralize endogenous peroxidases. The samples were boiled in RNAscope Target Retrieval Reagent for 15 min, followed by incubation in RNAscope Protease Plus for 30 min and probe hybridization for 2 h. The signal was amplified and visualized by using the Brown (DAB) Detection Kit. Tissues were counterstained with 50% Gill’s haematoxylin and visualized with standard bright-field microscopy. The original magnification was 20×.

ZIKV-specific antibody detection

Serum IgG antibodies against ZIKV were detected by ELISA. Briefly, polysorb enzyme-linked immunosorbent assay plates (Nunc, USA) were coated at 50 ng per well with ZIKV prME protein (Sino Biological, China) diluted in phosphate buffered saline (PBS) overnight at 4 °C. Plates were blocked in 5% (vol/vol) bovine serum albumin in PBS for 1 h at 37 °C. Serum samples were serially diluted in PBS and added to the wells, and the plates were incubated for 1 h at 37 °C. The plates were then washed with PBST five times to remove unbound antibody. After washing, a suitable concentration of guinea pig IgG antibody (Abcam, UK) was added to the well for 1 h at 37 °C, TMB substrate (Solarbio, China) was added to the wells, and the plate was incubated for 20 min at room temperature in darkness. Finally, 2.0 M H2SO4 was added to stop the reaction, and the absorbance was measured at 450 nm using a microplate reader (Beckman, USA). The ZIKV E-specific antibody titre was calculated based on the highest dilution that resulted in a value twofold greater than the absorption of the control serum, with a cut-off value of 0.05.

Measurement of cytokines and chemokines

The concentrations of cytokines and chemokines were measured in the serum collected from guinea pigs by a commercial Luminex immunoassay kit (Cytokine & Chemokine 22-Plex Rat ProcartaPlex™ Panel, Thermo Fisher Scientific, Germany) according to the manufacturer’s instructions.

RNA library construction and sequencing

Guinea pigs before or after infection (1, 3 or 7 dpi) as previously described were used for RNA-seq. Total RNA from lung tissues was extracted using TRIzol (Invitrogen, USA) and DNase I (NEB, USA), respectively. Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Clustering of the index-coded samples was performed on a cBot cluster generation system using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, California, USA) according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on the Illumina NovaSeq 6000 platform, and 150-bp paired-end reads were generated. After sequencing, a Perl script was used to filter the original data (raw data) to clean reads by removing contaminating reads with adapter sequences and low-quality reads. Clean reads were aligned to the Cavia porcellus genome (Cavpor3.0). The number of reads mapped to each gene in each sample was counted by featureCounts v1.5.0-p3, and the FPKM (transcripts per kilobase of exon model per million mapped reads) was then calculated to estimate the expression levels of genes in each sample.

Bioinformatic analyses

The DESeq2 R package was used for differential gene expression analysis. Genes with Padj ≤ 0.05 and |Log2FC | > 1 were identified as differentially expressed genes (DEGs). The DEGs identified were used as queries to search for enriched biological processes (Gene Ontology BP) and KEGG pathway enrichment using Metascape. Heatmaps of gene expression levels were constructed using the pheatmap package in R (https://cran.rstudio.com/web/packages/pheatmap/index.html).

Quantification and statistical analysis

All data were analysed with GraphPad Prism 8.0 software. Unless specified, the data are presented as the mean ± SEM in all experiments. Analysis of variance (ANOVA) or a t test was used to determine statistical significance among different groups (*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001; n.s., not significant).